Protein S (PS) is a vitamin K-dependent protein of 75,000 molecular weight with 635 amino acid residues. DiScipio et al., Biochem., 18:899 (1979); Lundwall et al., Proc. Natl. Acad. Sci. U.S.A., 83:6716-6720 (1986); Hoskins et al., Proc. Natl. Acad. Sci. U.S.A., 84:394-353 (1987). Human plasma contains 346 nM PS of which 62% is complexed with the .beta. chain subunit of complement protein, C4b binding protein (C4BP), and 38% is not complexed to C4BP and considered "free PS". Griffin et al., Blood, 79:3203-3211 (1992); Dahlback et al., Proc. Natl. Acad. Sci. U.S.A., 78:2512 (1981); Dahlback et al., J. Biol. Chem., 265:16082 (1990); and Nelson et al., Biochemistry, 30:2384 (1991).
PS exhibits anticoagulant activity in in vitro clotting assays. PS demonstrates anticoagulant cofactor activity for activated protein C (APC), an anticoagulant serine protease enzyme. Walker, J. Biol. Chem., 255:5221-5224 (1980); Harris et al., J. Biol. Chem., 260:2007 (1985); Stern et al., J. Biol. Chem., 261:713 (1986); Walker, J. Biol. Chem., 256:11128 (1981); and Solymoss et al., J. Biol. Chem., 263:14884 (1988). PS has also been shown to be an anticoagulant factor in the absence of APC as it can inhibit prothrombinase activity in assays free of APC, (Mitchell et al., Thromb. Haemost., 60:298-304, 1988), and binds to Factor Va or Factor Xa and functions as an anticoagulant without APC. Heeb et al., Circulation, 86:3238a, 1992; and Heeb et al., Circulation, 86:1040a, 1992. In plasma, PS reversibly associates with C4BP with high affinity (dissociation constant of about 1-5 nanomolar). Only free PS is active as an APC cofactor and it is widely accepted that the association of PS with C4BP is associated with loss of the anticoagulant activity of PS. Dahlback, J. Biol. Chem., 261:12022 (1986 ); and Taylor et al., Blood, 78:357-363 (1991). Therefore, C4BP is effectively an inhibitor of PS anticoagulant activity. The anticoagulant activity of PS can also be diminished or lost by cleavage at arginine residues within the so-called "thrombin-sensitive loop" comprising residues 46-75. Chang et al., Circulation, 86:3241a (1992).
PS is physiologically a very important antithrombotic factor since hereditary or acquired deficiencies of PS are associated with venous and arterial thrombotic disease. Allaart et al., Thromb. Haemost., 64:206 (1990); Sie et al., Thromb. Haemost., 62:1040 (1989); Engesser et al., Ann. Intern. Med., 106:677 (1987); Mannucci et al., Thromb. Haemost., 55:440 (1986); and Schwartz et al., Blood, 74:213 (1989). It is widely accepted that because only free PS has anticoagulant activity vitro, the level of free PS in blood in vivo is considered the only relevant physiologic anticoagulantly active species. A deficiency of free PS with a normal level of total PS has been described in some patients with thrombotic disease (Comp et al., Blood, 67:504, 1986), and it has been hypothesized that an acquired deficiency of free PS due to temporary elevations of C4BP in disseminated intravascular coagulation or in a wide variety of inflammatory conditions, e.g. systemic lupus erythematosus, may contribute to a hypercoagulable state. Taylor et al., Blood, 78:357-363 (1991); Heeb et al., Blood, 73:455 (1989); Comp et al., Blood, 66:348a (1985); D'Angelo et al., J. Clin. Invest., 81:1445 (1988); Boerger et al., Blood, 69:692 (1987); and D'Angelo et al., Ann. Intern. Med., 107:42 (1987). In addition, PS has been suggested to be important in metastasizing carcinoma and leukemias and therefore can be used therapeutically to inhibit cancer cell growth. Kemkes-Matthes, Clin. Invest., 70: 529-534 (1992).
Recently it was shown in an experimental primate animal model that elevations of C4BP exacerbate host response and convert a nonlethal dose of E. coli into a lethal dose. Taylor et al., Blood, 78:357-363 (1991). It was also shown that treatment of animals receiving excess PS with the C4BP did not suffer the lethal outcome or the hypercoagulable responses, thus showing that free PS which is not bound to C4BP may be a useful therapeutic agent for infection, inflammation and hypercoagulability. Taylor et al., Blood, 78:357-363 (1991). Furthermore, Schwarz et al., have described the use of plasma-derived PS in in vivo therapeutic methods for treating thromboses and thromboembolic complications. U.S. Pat. No. 5,143,901.
Forms of PS that have reduced affinity for C4BP would provide useful therapeutic agents since they could be administered without risk of loss of activity associated with binding to C4BP. A variant PS was described by Chang et al., Blood, 78:1099a (1991), referred to as the L-variant, in which the carboxy terminal amino acid residues corresponding to residues 583-635 of PS were deleted. The L-variant was shown to retain anticoagulant activity comparable to wild-type PS and to exhibit greatly reduced C4BP binding ability. However, L-variant PS is substantially different from native PS, and may therefore elicit immune responses to those differences upon in vivo administration.
No known recombinant mutants of PS exist which are highly homologous to wild-type PS and which exhibit reduced binding affinity for C4BP species which contain .beta. subunits. This invention provides novel (variant) forms of recombinant PS containing point mutations that have reduced affinity for C4BP, and thereby can exhibit increased serum half life.